Folate Metabolism in Mycobacterium tuberculosis Revisited: A Potential Drug Targe

重新审视结核分枝杆菌中的叶酸代谢：潜在的药物目标

• 批准号: 7862191
• 负责人: Liem Duy Nguyen
• 金额: $ 39.25万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862191
• 关键词: Antibiotic Resistance; Antibiotics; Antimycobacterial Agents; Antitubercular Agents; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Cell Wall; Chemicals; Clinical; Complement; Cyclic GMP-Dependent Protein Kinases; Development; Drug Resistant Tuberculosis; Drug resistance; Drug-sensitive; Effectiveness; Enzymes; Epidemic; Ethambutol; Extreme drug resistant tuberculosis; Folate; Folate Biosynthesis Pathway; Folic Acid Antagonists; GTP-Binding Proteins; Genes; Genetic; Genus Mycobacterium; Homologous Gene; Human; In Vitro; Interruption; Knowledge; Laboratories; Libraries; Metabolism; Mission; Molecular; Multi-Drug Resistance; Mycobacterium tuberculosis; Natural Resistance; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Predisposition; Proteins; Pterins; Regimen; Regulation; Research; Resistance; Rifampin; Role; Screening procedure; Testing; Tuberculosis; United States National Institutes of Health; cGMP-dependent protein kinase Ibeta; combat; combinatorial; design; drug development; drug efficacy; efficacy testing; enzyme activity; folic acid metabolism; genome-wide; improved; inhibitor/antagonist; insight; interest; isoniazid; macrophage; mutant; novel; novel strategies; novel therapeutic intervention; p-Aminosalicylic Acid; pre-clinical; public health relevance; pyrophosphatase; resistance mechanism; tripolyphosphate; tuberculosis drugs

DESCRIPTION (provided by applicant): The worldwide emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) is severely complicating the current tuberculosis (TB) epidemic. New TB drugs are urgently needed to combat MDR/XDR TB and to improve the current 6-month drug regimens for non-resistant TB. The folate biosynthetic pathway has been an attractive target for antibiotic development since it is absent in humans. A preliminary study in our laboratory using a transposon insertion library in M. smegmatis has identified several novel determinants of antifolate resistance in mycobacteria. One antifolate sensitive mutant encodes a homolog of the eukaryotic-type protein kinase G (PknG), recently identified as possible regulator of persistence of pathogenic mycobacteria in macrophages. Preliminary studies reveal that PknG regulates de novo folate biosynthesis by modulating the activity of a dihydroneopterin triphosphate pyrophosphatase that controls the influx of pterin moiety into the folate pathway. This novel regulatory mechanism has not been previously identified for de novo folate biosynthesis. Both genetic interruption and specific chemical inhibition of PknG kinase activity result in hyper-susceptibility of mycobacteria not only to antifolate drugs but also other antibiotics, including frontline TB drugs such as rifampicin and ethambutol. This is due to a direct effect on de novo folate biosynthesis and an indirect effect by altering cell wall permeability, respectively. The central hypothesis of this application is that genes defining intrinsic antifolate resistance encode proteins that can be targeted by potentiators that sensitize Mtb to antifolate drugs by inhibiting the resistance mechanisms. Specifically, pharmaceutical inactivation of PknG could sensitize Mtb to antifolates and multiple other approved drugs, to which it is currently resistant. Three specific aims are designed to test this hypothesis. First, using a non-biased approach, we will identify and characterize the entire genome-wide antifolate resistant determinants (the antifolate resistome) of Mtb. Secondly, we will rigorously investigate the molecular mechanisms of PknG-regulated folate-biosynthesis in Mtb. Lastly, we will characterize the potentiating effects of PknG inhibitors on antifolate drugs and the efficacy of their combined effect against drug-resistant and non-resistant Mtb. These proposed studies will not only provide insight into a previously unknown regulatory mechanism of de novo folate biosynthesis in bacteria but also into the mechanisms of intrinsic resistance of Mtb to antifolate drugs. In terms of drug development, these studies will reveal novel targets and provide proof of concept that inhibition of intrinsic resistance pathways in Mtb can be used to improve the effectiveness of already available antibiotics. ) PUBLIC HEALTH RELEVANCE: Because of its absence in humans, de novo folate biosynthesis provides an attractive target for development of novel antibiotics that help reduce the current epidemic of drug resistant bacterial infections, including the multidrug resistant and extensively drug resistant tuberculosis (MDR/XDR TB). Besides other targets, our research identified the eukaryotic-type protein kinase G (PknG) as a novel regulator that controls de novo folate biosynthesis in Mycobacterium tuberculosis, the causative agent of TB, by regulating activity of an enzyme that converts the pterin moiety for entry into the folate synthetic pathway. This regulatory control of folate biosynthesis is novel and could be targeted to potentiate anti-TB activity of antifolate drugs thus providing a new approach to the treatment for MDR/XDR TB; therefore our findings will be relevant to the mission of the NIH and will be of interest to both industrial and academic entities that are developing new drugs to combat bacterial antibiotic resistance.

描述（由申请人提供）：全球范围内出现的耐多药（MDR）和广泛耐药（XDR）结核分枝杆菌（Mtb）菌株使当前的结核病（TB）流行严重复杂化。迫切需要新的结核病药物来对抗耐多药/广泛耐药结核病并改进目前的非耐药结核病 6 个月药物治疗方案。叶酸生物合成途径一直是抗生素开发的一个有吸引力的目标，因为它在人类中不存在。我们实验室使用耻垢分枝杆菌中的转座子插入文库进行的初步研究已经确定了分枝杆菌中抗叶酸剂耐药性的几个新决定因素。一种抗叶酸敏感突变体编码真核型蛋白激酶 G (PknG) 的同源物，最近被鉴定为巨噬细胞中致病性分枝杆菌持续存在的可能调节因子。初步研究表明，PknG 通过调节二氢新蝶呤三磷酸焦磷酸酶的活性来调节叶酸从头生物合成，该焦磷酸酶控制蝶呤部分流入叶酸途径。此前尚未发现这种新的叶酸生物合成新调节机制。 PknG 激酶活性的基因中断和特异性化学抑制都会导致分枝杆菌不仅对抗叶酸药物高度敏感，而且对其他抗生素也高度敏感，包括利福平和乙胺丁醇等一线结核病药物。这是由于分别对叶酸从头生物合成的直接影响和通过改变细胞壁渗透性的间接影响。本申请的中心假设是，定义内在抗叶酸药物耐药性的基因编码的蛋白质可以被增效剂靶向，通过抑制耐药机制，使结核分枝杆菌对抗叶酸药物敏感。具体来说，PknG 的药物灭活可能会使 Mtb 对抗叶酸剂和多种其他已批准的药物敏感，而 Mtb 目前对这些药物具有耐药性。设计了三个具体目标来检验这一假设。首先，我们将使用无偏见的方法来识别和表征 Mtb 的整个基因组抗叶酸抗性决定簇（抗叶酸抗性组）。其次，我们将严格研究 Mtb 中 PknG 调节叶酸生物合成的分子机制。最后，我们将描述 PknG 抑制剂对抗叶酸药物的增强作用以及它们对耐药和非耐药 Mtb 的联合作用的功效。这些拟议的研究不仅将深入了解细菌中从头叶酸生物合成的先前未知的调节机制，而且还将深入了解结核分枝杆菌抗叶酸药物的内在耐药机制。在药物开发方面，这些研究将揭示新的靶标，并提供概念证明，即抑制结核分枝杆菌的内在耐药途径可用于提高现有抗生素的有效性。 ） 公共卫生相关性：由于叶酸在人体中不存在，因此叶酸从头生物合成为新型抗生素的开发提供了一个有吸引力的目标，有助于减少当前耐药细菌感染的流行，包括耐多药和广泛耐药结核病（MDR/XDR TB） ）。除了其他靶点外，我们的研究还发现真核型蛋白激酶 G (PknG) 是一种新型调节剂，通过调节将蝶呤部分转化为进入的酶的活性，控制结核分枝杆菌（结核病的病原体）中叶酸的从头生物合成。进入叶酸合成途径。这种叶酸生物合成的调节控制是新颖的，可以有针对性地增强抗叶酸药物的抗结核活性，从而为治疗耐多药/广泛耐药结核病提供一种新方法；因此，我们的研究结果将与美国国立卫生研究院的使命相关，并将引起正在开发新药来对抗细菌抗生素耐药性的工业和学术实体的兴趣。